Cx43-mediated sorting of miRNAs into extracellular vesicles.

Through the exchange of lipids, proteins, and nucleic acids, extracellular vesicles (EV) allow for cell-cell communication across distant cells and tissues to regulate a wide range of physiological and pathological processes. Although some molecular mediators have been discovered, the mechanisms underlying the selective sorting of miRNAs into EV remain elusive. Previous studies demonstrated that connexin43 (Cx43) forms functional channels at the EV surface, mediating the communication with recipient cells. Here, we show that Cx43 participates in the selective sorting of miRNAs into EV through a process that can also involve RNA-binding proteins. We provide evidence that Cx43 can directly bind to specific miRNAs, namely those containing stable secondary structure elements, including miR-133b. Furthermore, Cx43 facilitates the delivery of EV-miRNAs into recipient cells. Phenotypically, we show that Cx43-mediated EV-miRNAs sorting modulates autophagy. Overall, our study ascribes another biological role to Cx43, that is, the selective incorporation of miRNAs into EV, which potentially modulates multiple biological processes in target cells and may have implications for human health and disease.

Effects of neonatal exposure to methoxychlor on corpus luteum in gilts: A transcriptomic analysis.

This study investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities, on luteal function in pigs. Piglets were injected subcutaneously with MXC (20 µg/kg body weight) or corn oil (control) between postnatal Days 1 and 10 (N = 5/group). Corpora lutea from sexually mature gilts were examined for luteal steroid and prostaglandin concentrations and processed for total RNA isolation and subsequent RNA sequencing. Intra-luteal concentrations of androstenedione and prostaglandin E2 were greater, while that of estrone was lower when compared to control. Fifty-three differentially expressed (DE) microRNAS (miRNAs) (p-adjusted <.05 and log2(fold change) ≥.5) and 359 DE genes (p-adjusted <.05 and log2(fold change) ≥1) were identified in luteal tissue in response to neonatal MXC treatment. MXC was found to affect the expression of genes related to lipogenesis, steroidogenesis, membrane transport, immune response, cell signaling and adhesion. These results suggest an earlier onset of structural luteolysis in pigs caused by MXC actions in neonates. Since negative correlation analysis showed the potential interactions of miRNAs with specific messenger RNAs, we propose that these miRNAs are potential mediators of the long-term MXC effect on the CL function in pigs.

Association between miR-146a and Tumor Necrosis Factor Alpha (TNF-α) in Stable Coronary Artery Disease.

Background and Objectives: Tumor necrosis factor alpha (TNF-α) is proatherogenic and associated with the risk of acute ischemic events, although the mechanisms that regulate TNF-α expression in stable coronary artery disease (SCAD) are not fully understood. We investigated whether metabolic, inflammatory, and epigenetic (microRNA (miRNA)) markers are associated with TNF-α expression in SCAD. Materials and Methods: Patients with SCAD were prospectively recruited and their metabolic and inflammatory profiles were assessed. TNF-α levels were assessed using an enzyme-linked immunosorbent assay. The relative expression of six circulating miRNAs associated with the regulation of inflammation and/or atherosclerosis was determined. Results: Of the 24 included patients with the mean age of 65 (9) years, 88% were male, and 54% were diabetic. The TNF-α levels were (median (interquartile range)) 1.0 (0.7-1.1) pg/mL. The percentage of glycosylated hemoglobin (r = 0.418, p = 0.042), serum triglyceride levels (r = 0.429, p = 0.037), and C-reactive protein levels (r = 0.407, p = 0.048) were positively correlated with TNF-α levels. Of the candidate miRNAs, miR-146a expression levels were negatively correlated with TNF-α levels (as indicated by r = 0.500, p = 0.035 for correlation between delta cycle threshold (ΔCt) miR-146a and TNF-α levels). In multivariate analysis, serum triglyceride levels and miR-146a expression levels were independently associated with TNF-α levels. miR-146 expression levels were not associated with metabolic or other inflammatory parameters and were negatively correlated with the number of coronary vessels with obstructive disease (as indicated by r = 0.556, p = 0.017 for correlation between ΔCt miR-146a and number of diseased vessels). Conclusions: miR-146a expression levels were negatively correlated with TNF-α levels in patients with SCAD, irrespective of other metabolic or inflammatory markers, and with the severity of coronary artery disease. The results add to the knowledge on the role of miR-146a in TNF-α-based inflammation in SCAD and support future research on the potential therapeutic use of miR-146a in such a clinical scenario.

Bioinformatics Research Methodology of Non-coding RNAs in Cardiovascular Diseases.

The transcriptional complexity generated by the human genomic output is within the core of cell and organ physiology, but also could be in the origin of pathologies. In cardiovascular diseases, the role of specific families of RNA transcripts belonging to the group of the non-coding RNAs started to be unveiled in the last two decades. The knowledge of the functional rules and roles of non-coding RNAs in the context of cardiovascular diseases is an important factor to derive new diagnostic methods, but also to design targeted therapeutic strategies. The characterization and analysis of ncRNA function requires a deep knowledge of the regulatory mechanism of these RNA species that often relies on intricated interaction networks. The use of specific bioinformatic tools to interrogate biological data and to derive functional implications is particularly relevant and needs to be extended to the general practice of translational researchers. This chapter briefly summarizes the bioinformatic tools and strategies that could be used for the characterization and functional analysis of non-coding RNAs, with special emphasis in their applications to the cardiovascular field.

Interactions Among Regulatory Non-coding RNAs Involved in Cardiovascular Diseases.

Non-coding RNAs (ncRNAs) are important regulatory players in human cells that have been shown to modulate different cellular processes and biological functions through controlling gene expression, being also involved in pathological conditions such as cardiovascular diseases. Among them, long non-coding RNAs (lncRNAs) and circular (circRNAs) could act as competing endogenous RNAs (ceRNAs) sequestering other ncRNAs. This entangled network of interactions has been reported to trigger the decay of the targeted ncRNAs having important roles in gene regulation. Growing evidences have been demonstrated that the regulatory mechanism underlying the crosstalk between different ncRNA species, namely lncRNAs, circRNAs and miRNAs has also an important role in the pathophysiological processes of cardiovascular diseases. In this chapter, the main regulatory relationship among lncRNAs, circRNAs and miRNAs were summarized and their role in the control and development of cardiovascular diseases was highlighted.

Interspecies Communication in Holobionts by Non-Coding RNA Exchange.

Complex organisms are associations of different cells that coexist and collaborate creating a living consortium, the holobiont. The relationships between the holobiont members are essential for proper homeostasis of the organisms, and they are founded on the establishment of complex inter-connections between all the cells. Non-coding RNAs are regulatory molecules that can also act as communication signals between cells, being involved in either homeostasis or dysbiosis of the holobionts. Eukaryotic and prokaryotic cells can transmit signals via non-coding RNAs while using specific extracellular conveyors that travel to the target cell and can be translated into a regulatory response by dedicated molecular machinery. Within holobionts, non-coding RNA regulatory signaling is involved in symbiotic and pathogenic relationships among the cells. This review analyzes current knowledge regarding the role of non-coding RNAs in cell-to-cell communication, with a special focus on the signaling between cells in multi-organism consortia.

Circular RNA-Centered Regulatory Networks in the Physiopathology of Cardiovascular Diseases.

Non-coding regulatory RNAs are generated as a core output of the eukaryotic genomes, being essential players in cell biology. At the organism level, they are key functional actors in those tissues and organs with limited proliferation capabilities such as the heart. The role of regulatory networks mediated by non-coding RNAs in the pathophysiology of cardiovascular conditions is starting to be unveiled. However, a deeper knowledge of the functional interactions among the diverse non-coding RNA families and their phenotypic consequences is required. This review presents the current knowledge about the functional crosstalk between circRNAs and other biomolecules in the framework of the cardiovascular diseases.

The circulating non-coding RNA landscape for biomarker research: lessons and prospects from cardiovascular diseases.

Pervasive transcription of the human genome is responsible for the production of a myriad of non-coding RNA molecules (ncRNAs) some of them with regulatory functions. The pivotal role of ncRNAs in cardiovascular biology has been unveiled in the last decade, starting from the characterization of the involvement of micro-RNAs in cardiovascular development and function, and followed by the use of circulating ncRNAs as biomarkers of cardiovascular diseases. The human non-coding secretome is composed by several RNA species that circulate in body fluids and could be used as biomarkers for diagnosis and outcome prediction. In cardiovascular diseases, secreted ncRNAs have been described as biomarkers of several conditions including myocardial infarction, cardiac failure, and atrial fibrillation. Among circulating ncRNAs, micro-RNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) have been proposed as biomarkers in different cardiovascular diseases. In comparison with standard biomarkers, the biochemical nature of ncRNAs offers better stability and flexible storage conditions of the samples, and increased sensitivity and specificity. In this review we describe the current trends and future prospects of the use of the ncRNA secretome components as biomarkers of cardiovascular diseases, including the opening questions related with their secretion mechanisms and regulatory actions.

Noncoding Transcriptional Landscape in Human Aging.

Aging is a universal phenomenon in metazoans, characterized by a general decline of the organism physiology associated with an increased risk of mortality and morbidity. Aging of an organism correlates with a decline in function of its cells, as shown for muscle, immune, and neuronal cells. As the DNA content of most cells within an organism remains largely identical throughout the life span, age-associated transcriptional changes must be achieved by epigenetic mechanisms. However, how aging may impact on the epigenetic state of cells is only beginning to be understood. In light of a growing number of studies demonstrating that noncoding RNAs can provide molecular signals that regulate expression of protein-coding genes and define epigenetic states of cells, we hypothesize that noncoding RNAs could play a direct role in inducing age-associated profiles of gene expression. In this context, the role of long noncoding RNAs (lncRNAs) as regulators of gene expression might be important for the overall transcriptional landscape observed in aged human cells. The possible functions of lncRNAs and other noncoding RNAs, and their roles in the regulation of aging-related cellular pathways will be analyzed.

Circulating miR-122-5p/miR-133b Ratio Is a Specific Early Prognostic Biomarker in Acute Myocardial Infarction.

BACKGROUND: MicroRNAs (miRNAs) are key players in cardiovascular development and disease. However, not only miRNAs of a cardiac origin have a critical role in heart function. Recent studies have demonstrated that miR-122-5p, a hepatic miRNA, increases in the bloodstream during ischemic cardiogenic shock and it is upregulated in the infarcted myocardium. The aim of the present study was to determine the potential of circulating miR-122-5p as a biomarker for early prognostic stratification of ST-segment elevation acute myocardial infarction (STEMI) patients. METHODS AND RESULTS: One hundred and forty-two consecutive STEMI patients treated with primary angioplasty were included in the study. Serum levels of miR-1-3p, -122-5p, -133a-3p, -133b, -208b-3p and -499a-5p were measured at the time of cardiac catheterization by quantitative polymerase chain reaction and related to in-hospital and long-term outcome. During a follow up of 20.8 months, 9 patients died, 6 had recurrence of myocardial infarction, and 26 patients suffered an adverse cardiovascular event. Event-free survival was significantly worse in patients with a higher miR-122-5p/133b ratio (3rd tertile distribution, above 1.42 Log(10)), having almost a 9-fold higher risk of death or myocardial infarction and a 4-fold higher risk of adverse cardiovascular events. CONCLUSIONS: This study showed that the miR-122-5p/133b ratio is a new prognostic biomarker for the early identification of STEMI patients at a higher risk of developing major adverse events after undergoing primary percutaneous coronary intervention. (Circ J 2016; 80: 2183-2191).

Unzippers, resolvers and sensors: a structural and functional biochemistry tale of RNA helicases.

The centrality of RNA within the biological world is an irrefutable fact that currently attracts increasing attention from the scientific community. The panoply of functional RNAs requires the existence of specific biological caretakers, RNA helicases, devoted to maintain the proper folding of those molecules, resolving unstable structures. However, evolution has taken advantage of the specific position and characteristics of RNA helicases to develop new functions for these proteins, which are at the interface of the basic processes for transference of information from DNA to proteins. RNA helicases are involved in many biologically relevant processes, not only as RNA chaperones, but also as signal transducers, scaffolds of molecular complexes, and regulatory elements. Structural biology studies during the last decade, founded in X-ray crystallography, have characterized in detail several RNA-helicases. This comprehensive review summarizes the structural knowledge accumulated in the last two decades within this family of proteins, with special emphasis on the structure-function relationships of the most widely-studied families of RNA helicases: the DEAD-box, RIG-I-like and viral NS3 classes.

Role of myocardial microRNAs in the long-term ventricular remodelling of patients with aortic stenosis.

Aims: We hypothesize that miRs are key players in the dynamics of the hypertrophy phenotype in aortic stenosis (AS) patients. In our study, we aimed to identify the transcriptional patterns (protein-coding transcripts and miRs) from myocardial sample biopsies that could be associated with the absence of left ventricular (LV) mass regression after aortic valve replacement (AVR) in patients with severe AS and LV hypertrophy. Methods and results: We prospectively included 40 patients with severe AS, LV hypertrophy, and preserved ejection fraction undergoing AVR. Myocardial biopsies obtained during surgery were analysed for transcriptomic analysis performed by next-generation sequencing. At a 1-year follow-up, no hypertrophy reversal was observed in about half of the patients in the absence of patient-prosthesis mismatch and prosthesis dysfunction of uncontrolled hypertension. Predictors of mass regression were assessed from clinical, echocardiographic, and biochemical variables as well as from 300 miRs obtained from myocardial specimens, allowing the identification 29 differentially expressed. miR-4709-3p was found as a positive independent predictor of hypertrophy regression together with high-sensitivity troponin T (cTNT-hs) as a negative predictor. Gene transcripts RFX1, SIX5, MAPK8IF3, and PKD1 were predicted as simultaneous targets of five upregulated miRs suggesting its importance in LV hypertrophy. Conclusion: In our cohort, tissue miR-4709-3p and cTNT-hs were independent predictors of hypertrophy regression. The hypertrophy reversal process will likely depend from a complex network where miRNAs may have an important role, allowing a potential opportunity for therapy.

Biogenesis and mechanism of action of small non-coding RNAs: insights from the point of view of structural biology.

Non-coding RNAs are dominant in the genomic output of the higher organisms being not simply occasional transcripts with idiosyncratic functions, but constituting an extensive regulatory network. Among all the species of non-coding RNAs, small non-coding RNAs (miRNAs, siRNAs and piRNAs) have been shown to be in the core of the regulatory machinery of all the genomic output in eukaryotic cells. Small non-coding RNAs are produced by several pathways containing specialized enzymes that process RNA transcripts. The mechanism of action of these molecules is also ensured by a group of effector proteins that are commonly engaged within high molecular weight protein-RNA complexes. In the last decade, the contribution of structural biology has been essential to the dissection of the molecular mechanisms involved in the biosynthesis and function of small non-coding RNAs.

Cigarette Smoking, miR-27b Downregulation, and Peripheral Artery Disease: Insights into the Mechanisms of Smoking Toxicity.

Cigarette smoking is a risk factor for the development of peripheral artery disease (PAD), although the proatherosclerotic mediators of cigarette smoking are not entirely known. We explored whether circulating microRNAs (miRNAs) are dysregulated in cigarette smokers and associated with the presence of PAD. Ninety-four participants were recruited, including 58 individuals without and 36 with PAD, 51 never smokers, 28 prior smokers, and 15 active smokers. The relative expression of six circulating miRNAs with distinct biological roles (miR-21, miR-27b, miR-29a, miR-126, miR-146, and miR-218) was assessed. Cigarette smoking was associated with the presence of PAD in multivariate analysis. Active smokers, but not prior smokers, presented miR-27b downregulation and higher leukocyte, neutrophil, and lymphocyte counts; miR-27b expression levels were independently associated with active smoking. Considering the metabolic and/or inflammatory abnormalities induced by cigarette smoking, miR-27b was independently associated with the presence of PAD and downregulated in patients with more extensive PAD. In conclusion, the atheroprotective miR-27b was downregulated in active smokers, but not in prior smokers, and miR-27b expression was independently associated with the presence of PAD. These unreported data suggest that the proatherogenic properties of cigarette smoking are mediated by a downregulation of miR-27b, which may be attenuated by smoking cessation.

Circulating miRNAs Are Associated with the Systemic Extent of Atherosclerosis: Novel Observations for miR-27b and miR-146.

The mechanisms that regulate the systemic extent of atherosclerosis are not fully understood. We investigated whether the expression of circulating miRNAs is associated with the extent of stable atherosclerosis to a single territory or multiple territories (polyvascular) and with the severity of atherosclerosis in each territory. Ninety-four participants were prospectively recruited and divided into five age- and sex-matched groups: presenting no atherosclerosis, isolated coronary atherosclerosis, coronary and lower extremity atherosclerosis, coronary and carotid atherosclerosis, and atherosclerosis of the coronary, lower extremity, and carotid territories. The expression of six circulating miRNAs with distinct biological roles was assessed. The expression of miR-27b and miR-146 differed across groups (p < 0.05), showing a decrease in the presence of atherosclerosis, particularly in the three territories. miR-27b and miR-146 expression decreased in association with a higher severity of coronary, lower extremity, and carotid atherosclerosis. Polyvascular atherosclerosis involving the three territories was independently associated with a decreased miR-27b and miR-146 expression. Both miRNAs presented an area under the curve of ≥0.75 for predicting polyvascular atherosclerosis involving the three territories. To conclude, miR-27b and miR-146 were associated with the presence of severe polyvascular atherosclerosis and with the atherosclerosis severity in each territory. Both are potential biomarkers of severe systemic atherosclerosis.

Circulating circRNA as biomarkers for dilated cardiomyopathy etiology.

Dilated cardiomyopathy (DCM) is the third most common cause of heart failure. The multidisciplinary nature of testing - involving genetics, imaging, or cardiovascular techniques - makes its diagnosis challenging. Novel and reliable biomarkers are needed for early identification and tailored personalized management. Peripheral circular RNAs (circRNAs), a leading research topic, remain mostly unexplored in DCM. We aimed to assess whether peripheral circRNAs are expressed differentially among etiology-based DCM. The study was based on a case-control multicentric study. We enrolled 130 subjects: healthy controls (n = 20), idiopathic DCM (n = 30), ischemic DCM (n = 20), and familial DCM patients which included pathogen variants of (i) LMNA gene (n = 30) and (ii) BCL2-associated athanogene 3 (BAG3) gene (n = 30). Differentially expressed circRNAs were analyzed in plasma samples by quantitative RT-PCR and correlated to relevant systolic and diastolic parameters. The pathophysiological implications were explored through bioinformatics tools. Four circRNAs were overexpressed compared to controls: hsa_circ_0003258, hsa_circ_0051238, and hsa_circ_0051239 in LMNA-related DCM and hsa_circ_0089762 in the ischemic DCM cohort. The obtained areas under the curve confirm the discriminative capacity of circRNAs. The circRNAs correlated with some diastolic and systolic echocardiographic parameters with notable diagnostic potential in DCM. Circulating circRNAs may be helpful for the etiology-based diagnosis of DCM as a non-invasive biomarker. KEY MESSAGES: The limitations of cardiac diagnostic imaging and the absence of a robust biomarker reveal the need for a diagnostic tool for dilated cardiomyopathy (DCM). The circular RNA (circRNA) expression pattern is paramount for categorizing the DCM etiologies. Our peripheral circRNAs fingerprint discriminates between various among etiology-based DCM and correlates with some echocardiographic parameters. We provide a potential non-invasive biomarker for the etiology-based diagnosis of LMNA-related DCM and ischemic DCM.

A transcriptome approach evaluating effects of neonatal androgen and anti-androgen treatments on regulation of luteal function in sexually mature pigs.

The current study was designed to gain insights into regulatory mechanisms mediating long-term effects of androgen excess or deficiency on corpus luteum function in pigs. Piglets were injected subcutaneously with testosterone propionate (TP, an androgen), flutamide (FLU, an anti-androgen) or corn oil (control) between postnatal Days 1 and 10. Corpora lutea from sexually mature gilts were examined for luteal steroid concentrations and processed for total RNA isolation and subsequent RNA sequencing to determine abundances of mRNA transcripts and microRNAs (miRNAs). Potential miRNA-mRNA interactions were explored in silico. Androstenedione, testosterone and estrone concentrations in corpora lutea were altered due to the disrupted androgen action in neonates. The luteal tissue had 465 and 353 genes for which there were differential mRNA abundances as compared with the control group (P-adjusted < 0.05; log2FC ≥ 1.0) in response to neonatal TP and FLU piglet treatments, respectively. Disruption of androgen signalling in neonates affected mRNA transcript abundance, as compared with the control group, for genes associated with apoptosis, angiogenesis and immune functions in the corpora lutea. Furthermore, there was a differential abundance of a group of miRNAs in the treatment groups compared with the control group. These results indicate the neonatal androgenic milieu affects the onset of luteolysis when these animals are sexually mature, although mechanisms for responses to TP or FLU likely differ. It is proposed that changes in specific miRNAs and mRNAs may, in part, account for long-term effects of androgen excess or androgen deficiency on corpus luteum function in pigs.

New LncRNAs in Chronic Hepatitis C progression: from fibrosis to hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death in the world, and about 80% of the cases are associated with hepatitis B or C. Genetic and epigenetic alterations are accumulated over decades of chronic injury and may affect the functioning of tumor suppressor genes and protooncogenes. Studies have evidenced the role of Long non-coding RNAs (LncRNA) with oncogenic or tumor suppressor activities, suggesting a great potential in the treatment, diagnosis or indicator of prognosis in cancer. In this context, the aim of this study was to evaluate the global expression profile lncRNA in hepatic tissue samples with different stages of fibrosis associated with chronic hepatitis C, HCC and normal liver, in order to identify new lncRNAs that could contribute to study the progression of hepatic fibrosis to HCC associated with chronic hepatitis C. RNA-Seq was performed on Illumina NextSeq platform to identify lncRNAs expressed differently in 15 patients with chronic hepatitis C, three patients with HCC and three normal liver specimens. When the pathological tissues (fibrosis and carcinoma) were compared to normal hepatic tissue, were identified 2, 6 e 34 differentially expressed lncRNAs in moderate fibrosis, advanced fibrosis and HCC, respectively. The carcinoma group had the highest proportion of differentially expressed lncRNA (34) and of these, 29 were exclusive in this type of tissue. A heat map of the deregulated lncRNA revealed different expression patterns along the progression of fibrosis to HCC. The results showed the deregulation of some lncRNA already classified as tumor suppressors in HCC and other cancers, as well as some unpublished lncRNA whose function is unknown. Some of these lncRNAs are dysregulated since the early stages of liver injury in patients with hepatitis C, others overexpressed only in tumor tissue, indicating themselves as candidates of markers of fibrosis progression or tumor, with potential clinical applications in prognosis as well as a therapeutic target. Although there are already studies on lncRNA in hepatocellular carcinoma, this is the first study conducted in samples exclusively of HCV-related liver and HCV HCC.

Si vis pacem para bellum: A prospective in silico analysis of miRNA-based plant defenses against fungal infections.

Fungal pathogens are an important threat for plant crops, being responsible for important reductions of production yields and a consequent economic impact. Among the molecular mediators of fungal infections of plant crops, non-coding RNAs (ncRNAs) have been described as relevant players either in the plant immune responses and mechanism of defense or in the colonization of plant tissues by fungi. Acting as a mechanism of defense, some plant small ncRNAs such as miRNAs and tasiRNAs can be secreted by cells and directed to target the transcriptome of pathogenic fungi, triggering an RNAi-like interference mechanism able to silence the expression of specific fungal genes. The detailed knowledge of this mechanism of defense against fungal pathogens could open new possibilities for the protection of human important crops. To infer putative functional relationships mediated by ncRNA communication, we performed a prospective analysis to determine potential plant miRNAs able to target the genome of fungal pathogens, which resulted in the description of enriched specific plant miRNA families and their putative fungal targets that could be further studied in the context of plant-fungi interactions. The expression profile of specific members of the enriched miRNAs families showed an infection-dependent behavior in laboratory models of infection. Plant miRNAs showed sequence complementarity with coding genes of their cognate fungal pathogens. Plant miRNAs could potentially target fungal genes belonging to functional families related to stress response, membrane architecture, vacuolar transport, membrane traffic, and anabolic processes. Families of specific infection-responsive miRNAs are included in the putative plant defense mechanism.